Tuberculosis kills between 1.5-2 million people globally every year. A key defense against Mycobacterium tuberculosis (Mtb) infections is the production of nitric oxide (NO) by macrophages. Although NO controls Mtb growth, it rarely sterilizes the bacterium from the host. Therefore, we reasoned that Mtb has mechanisms to resist NO toxicity. We identified two genes in Mtb, mpa (Mycobacterium proteasome ATPase) and pafA (proteasome associated factor), that are required for protection against NO. Importantly, mpa and pafA mutants are severely attenuated in mice for reasons in addition to NO sensitivity. The proteasome is a multi-subunit, barrel shaped complex that degrades proteins. We hypothesize that Mpa and PafA chaperone proteins into the Mtb proteasome for degradation, but we do not know why this activity protects Mtb against NO or promotes virulence in mice. Although Mpa and PafA appear to function with the Mtb proteasome, this remains to be determined conclusively. Thus, the goals of this proposal are to determine if Mpa and PafA work with the proteasome to promote protein degradation, and understand why this activity protects Mtb against NO toxicity and promotes bacterial growth in mice. We will identify proteins that interact with Mpa in order to reconstitute degradation of native proteins in vitro. We will also identify substrates of the proteasome; this may help us understand proteasome activity is important for NOresistance and virulence. Finally, we will examine proteasome-dependent gene expression. It is possible that genes regulated by the proteasome are required for NO-resistance or pathogenesis. We will isolate mutants or regulated genes and assess their survival in NO and in mice. Relevance to public health: With increased age expectancy, prevalence of HIV infections and number of persons taking immunosuppressive drugs, the chances of contracting tuberculosis increases. Furthermore, tuberculosis therapy takes 6-9 months, a problem that leads to decreased compliance for taking antibiotics and increased chances of developing drug-resistant strains of Mtb. Taken together it will be important to develop faster acting drugs to new targets in Mtb in order to better treat tuberculosis in the future.